Construction and Bioinformatics Analysis of TRPV4 Prokaryotic Expression Purification System

Objective: This study aimed to construct the prokaryotic expression and purification system of non-selective cation channel TRPV4 protein and to carry out bioinformatics analysis to provide technical support for the study of diseases related to the target protein and the screening of drug active ingredients. Methods: The human TRPV4 gene was cloned into pET-28a(+), pET-32a, pET-15b, pGEX-5X-1, pEX-4T and pGEX-6p-1, respectively, and the prokaryotic expression system of TRPV4 was constructed using two E.coli expression hosts, BL21 and Rossetta. The target protein was isolated and purified by glutathione affinity chromatography and nickel column affinity chromatography. The target protein was analyzed by bioinformatics technology to obtain its corresponding physical, chemical properties and structural parameters. Results: The prokaryotic expression system of GST-TRPV4 and GST-TRPV4-6his fusion proteins was constructed using pGEX-6p-1 vector and Rossetta. The GST-TRPV4 fusion protein was significantly expressed when the IPTG concentrationwas 0.6 mmol/L, and the GST-TRPV4-6his fusion protein was significantly expressed when the IPTG concentration was 0.4 mmol/L, and the expression temperature was 18 ℃. When GST-TRPV4-6his fusion protein was purified, the complete GST-TRPV4-6his fusion protein could be obtained by imidazole solution at 100 mmol/L or 200 mmol/L. Conclusion: In this paper, human TRPV4 ion channel protein was successfully constructed, expressed and purified in prokaryotic expression system for the first time, and the physicochemical properties of the protein were analyzed by bioinformatics, which laid a good foundation for subsequent high-purity mass expression and further study.